ABSTRACT The tumor necrosis factor receptors (TNFRs) are a superfamily of transmembrane proteins that play critical roles in apoptosis and inflammatory diseases and are considered important therapeutic targets. Even though targeting of TNFRs is a billion-dollar industry, the clinically available drugs cause devastating side effects because they lack receptor specificity. My research focuses on understanding the essential conformational dynamics of TNFRs that transduce signals across the membrane, with the ultimate goal of enabling highly effective and specific targeting. To accelerate scientific discovery, we have focused on two of the most clinically relevant members of the superfamily: TNFR1, involved in various autoimmune diseases, including rheumatoid arthritis; and Death Receptor 5, one of the most actively pursued anti-cancer targets. We apply an investigative strategy that includes computational molecular modeling, thermodynamic calculations, and in vitro experimental tools, enabling us to predict and understand conformational changes in these single-pass transmembrane proteins. Our work has yielded important findings published in high-impact journals. For instance, we elucidated mechanisms of ligand binding in both TNFR1 and DR5. We found that binding is controlled by an interaction between methionine and aromatic amino acids, causing a conformational rearrangement of the ligand-binding pocket. Our studies of this interaction motif led to a fundamental discovery that answered a long-standing question regarding the role of methionine in protein folding, and further, how methionine oxidation causes protein misfolding. We built a new model of TNFR oligomerization that led us to discover that ligand binding causes a large-scale backbone conformational change in the extracellular domain of the receptor. This finding revised previous assumptions regarding TNFRs that activation occurs without any conformational changes in the receptor backbone. With computation and biophysical and cellular experiments, we also showed for the first time a scissors-like opening that occurs in the transmembrane domain helices and explained the fundamental thermodynamics of this process. Significantly, using FRET-based small molecule discovery, we built on our new model of TNFR activation and showed that allosteric alteration of the conformational states of TNFRs can inhibit activation, and have thereby opened new avenues to therapeutic intervention. We propose to extend our discoveries by integrating the dynamic modes across domains of the receptor and answering the fundamental question: what is the structural and dynamic mechanism of TNFR activation? We will address impactful questions, some of which may be high-risk, but with potential to be transformative in the field and to launch new directions in drug discovery. Our productivity is enhanced by longstanding interdisciplinary collaborations that engage additional biophysical tools, including EPR and NMR. The MIRA grant will provide flexibility to methodically, and deeply, address fundamental questions regarding TNFR signaling, which will profoundly enhance efforts to identify and rationally target the most vulnerable structural motifs in these important proteins.!